CN114164189A - Nicotinamide phosphoribosyl transferase mutant - Google Patents
Nicotinamide phosphoribosyl transferase mutant Download PDFInfo
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- CN114164189A CN114164189A CN202010945768.1A CN202010945768A CN114164189A CN 114164189 A CN114164189 A CN 114164189A CN 202010945768 A CN202010945768 A CN 202010945768A CN 114164189 A CN114164189 A CN 114164189A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02012—Nicotinamide phosphoribosyltransferase (2.4.2.12), i.e. visfatin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a nicotinamide phosphoribosyl transferase mutant, which can convert nicotinamide and phosphoribosyl pyrophosphate into beta-nicotinamide mononucleotide (beta-NMN). Compared with the wild type, the half-life period of the mutant is improved by 5.3 times, the storage time and the conversion efficiency of the nicotinamide phosphoribosyl transferase are improved, and the mutant is more suitable for industrial production and application.
Description
The technical field is as follows:
the invention belongs to the technical field of protein engineering, and particularly relates to a nicotinamide phosphoribosyltransferase mutant for preparing beta-nicotinamide mononucleotide.
Background art:
the Nicotinamide Mononucleotide is also called beta-Nicotinamide Mononucleotide (beta-Nicotinamide mononuleotide, beta-NMN), and has a structure shown as a compound I.
Beta-nicotinamide mononucleotide plays an important role in the production of energy in human cells and is involved in intracellular NAD+(nicotinamide adenine dinucleotide, an important coenzyme for cellular energy conversion) is NAD+Is one of the key precursors of (a). The David Scinclair research team reported in the literature Science,2017,355,1312-1317 NAD+The increase in the bodies of the mice can delay the aging signs of tissues and muscles of the aged mice, and the research work makes human beings greatly step towards realizing the longevity dream. NAD (nicotinamide adenine dinucleotide)+The molecular weight is too large, and the oral absorption and utilization rate is low. But with the addition of NAD+Research finding of precursor small molecular substance beta-NMNThe administration of beta-NMN can effectively promote in vivo NAD+And remarkably improves the metabolism of a human body, so that the beta-NMN becomes a medicine for preventing the aging. This also encourages researchers in the world of medicine, food, and cosmetics to continuously research and develop beta-NMN.
At present, in the biological enzyme synthesis of the beta-NMN, nicotinamide phosphoribosyl transferase (NAMPT) is the most key rate-limiting enzyme, and can catalyze the reaction of nicotinamide and phosphoribosyl pyrophosphate to synthesize the beta-NMN, and the conversion reaction formula is shown as Scheme 1.
In the catalytic reaction, the catalytic efficiency and stability of the enzyme are important indexes of industrial application, while the existing wild nicotinamide phosphoribosyl transferase is poor in catalytic efficiency and stability, and the industrial application of the beta-NMN biocatalysis technology is severely restricted.
Therefore, the improvement of the stability of the nicotinamide phosphoribosyltransferase is a key factor for reducing the biocatalytic synthesis cost of the beta-NMN, improving the industrial application value of the nicotinamide phosphoribosyltransferase and promoting the application of a biocatalytic technology in the industrial production of the beta-NMN.
The invention content is as follows:
aiming at the problems of low stability and low industrial application value of nicotinamide phosphoribosyl transferase in the prior art, the invention aims to provide a nicotinamide phosphoribosyl transferase mutant with higher stability than that of the existing wild type nicotinamide phosphoribosyl transferase mutant.
In one aspect, the invention provides a nicotinamide phosphoribosyltransferase mutant, which takes the amino acid sequence of the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO.1 as a reference sequence and has single-point or multi-point mutation at positions 314, 315, 417, 419, 450 and 452. Wherein Ser at position 314 is mutated into Cys, Gly at position 315 is mutated into Cys, Pro at position 417 is mutated into Cys, Ala at position 419 is mutated into Cys, Leu at position 450 is mutated into Pro, and Glu at position 452 is mutated into Pro.
Further, the amino acid sequence of the nicotinamide phosphoribosyltransferase mutant is shown in SEQ ID No.3, 5, 7, 9, 11, 13 and 15.
Further, the nucleotide sequence of the nicotinamide phosphoribosyltransferase mutant is shown in SEQ ID NO.4, 6, 8, 10, 12, 14 and 16.
Further, the nicotinamide phosphoribosyltransferase mutant is derived from Homo sapiens, and the accession number of the wild-type template NCBI is NM-005746.3.
Further, the nicotinamide phosphoribosyltransferase mutant is expressed in escherichia coli e.coli BL (21) DE 3.
Further, the expression vector of the nicotinamide phosphoribosyltransferase mutant is pET28 a.
In another aspect, the present invention provides the use of a nicotinamide phosphoribosyltransferase mutant, which can be used in a process for preparing nicotinamide mononucleotide, catalyzing the conversion of nicotinamide and phosphoribosyl pyrophosphate into nicotinamide mononucleotide.
The invention has the beneficial effects that: the invention provides a nicotinamide phosphoribosyltransferase mutant, which is characterized in that site-directed mutagenesis is carried out on a nicotinamide phosphoribosyltransferase gene sequence to finally obtain the nicotinamide phosphoribosyltransferase mutant with high stability, the mutant can efficiently catalyze nicotinamide and phosphoribosyl pyrophosphate to be converted into beta-NMN, the cost for producing the beta-NMN by applying a biological catalysis technology in industry is greatly reduced, and the nicotinamide phosphoribosyltransferase mutant has high industrial application value.
Drawings
FIG. 1 electrophoretogram of NAMPT gene
FIG. 2 protein expression profile of wild type NAMPT
Detailed Description
The technical content of the present invention is further described below with reference to specific examples for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
EXAMPLE 1 cloning construction of wild-type NAMPT
NAMPT gene is amplified by designing an upstream primer and a downstream primer, wherein the upstream primer comprises the following components: 5'-CGATCGCATATGAATCCTGCGGCAGAA-3', the underlined part is the restriction site of restriction enzyme Nde I; a downstream primer: 5'-ATGCTAGGAATTCCTAATGATG TG CTGCTTCCAG-3', the underlined part is the restriction site of the restriction enzyme EcoRI.
The target gene NAMPT was amplified with the help of primers from upstream and downstream using Prime STAR polymerase (TaKaRa). The PCR reaction conditions are as follows: pre-denaturation at 98 deg.C for 2min, each cycle comprising denaturation at 95 deg.C for 25s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 2min for 30 cycles; finally derivatization at 72 ℃ for 5 min. After the reaction, the PCR product was detected on a 1% agarose gel, and the results are shown in FIG. 1. The nucleic acid length is consistent with the published size of PDB database (1473bp), and the nucleotide sequence is shown as SEQ ID No. 2. The product was purified using a kit (Axygen), the purified product was digested simultaneously with restriction enzymes Nde I and EcoR I (Thermo), the digested gene and plasmid fragment were ligated using T40ligase, the ligated product was purified again and directly transformed into E.coli DH 5. alpha. competent cells, incubated at 37 ℃ for 1h, and directly spread on an LB plate containing 25. mu.g/ml kanamycin resistance, to obtain a recombinant plasmid designated pET-28 a-NAMPT.
Example 2 protein expression of wild-type NAMPT
The constructed recombinant plasmid pET28a-NAMPT is transformed into BL21(DE3) competent cells by a chemical transformation method, and the competent cells are cultured in a culture medium containing Kan+Resistant LB plates were cultured overnight in an inverted culture at 37 ℃. Selecting positive monoclonal cells to obtain the nicotinamide phosphoribosyltransferase gene engineering strain capable of inducing expression. BL21(DE3) cells containing NAMPT gene were inoculated into cells containing Kan+The mixture was cultured overnight at 37 ℃ in a resistant LB tube to obtain a primary seed culture. Transferring the seed culture solution into a 2YT liquid culture medium containing resistance according to the inoculation ratio of 1%, placing the culture medium in a shaking table, culturing for 3-5h at 37 ℃ and 200rpm, cooling to 20 ℃ when OD reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) with the concentration controlled at 0.1mM, and performing overnight induction expression. The fermentation broth was centrifuged to collect the cells, and the collected cells were washed with 20mM phosphate buffer (pH7.5)The cells were lysed and the cells were disrupted by sonication. Then, the mixture is centrifuged at 12000rpm for 10min again, and the obtained supernatant is the transaminase protein, and the expression condition of the protein can be seen through electrophoresis, which is shown in figure 2.
EXAMPLE 3 construction of mutants
Site-directed mutagenesis was performed on positions 314, 315, 417, 419, 450 and 452, respectively, according to the crystal 3DHD structure of NAMPT, using Primer5 software to design mutation primers, and using whole-plasmid PCR to obtain mutants, the specific Primer design is shown in table 1.
TABLE 1 primer sequences for site-directed mutagenesis
The underlined sequences in Table 1 were used as mutation sites, and the PCR reaction was carried out using Primer STAR max DNA polymerase (TaKaRa) in the presence of forward and reverse saturation primers, using recombinant plasmid pET28a-NAMPT as a template, and the PCR reaction program was as shown in Table 2.
TABLE 2 Whole plasmid amplification schedule
The obtained whole plasmid mutant fragment gene was transferred into BL21(DE3) competent cells and plated on Kan cells containing 30ug/mL+And (3) culturing the strain on a resistant LB plate at 37 ℃ overnight, and selecting a single clone to perform sequencing to identify whether the site is mutated. After obtaining mutants at a single point, analyzing the stability change respectively, and superposing the mutants with improved stability for mutation again, wherein the mutation method is the same as the above method.
Example 4 comparison of the stability of the mutants
In order to test whether the stability of the mutant is improved, firstly, the mutant obtained by the genetic engineering means is subjected to shake flask expression cell, the obtained cell is placed in an ultralow temperature refrigerator at minus 80 ℃ for freezing and crushing, after the cell is placed for 2 days, a plurality of 0.5g of crushed cells are respectively weighed, transferred into an EP (EP) tube and placed in a water pan at 25 ℃. Sampling, converting and testing participation activities in different time periods, wherein the specific feeding is as follows: preparing 50mM phosphoribosyl pyrophosphate (PRPP) by using 20mM phosphate buffer solution with pH of 7.5, putting 10mL of PPRP into a reaction kettle, weighing 0.061g of Nicotinamide (NAM) and transferring into the reaction kettle, putting 0.5g of NAMPT mutant which is thermally inactivated in different time periods into the reaction kettle, starting timing reaction at 37 ℃, heating the reaction solution to 90 ℃ after 4 hours, keeping for 5 minutes and stopping the reaction. The reaction was centrifuged at 12000rpm for 2min and analyzed by HPLC for product formation, with specific stability half-lives as shown in Table 3.
TABLE 3 stability data for NAMPT mutants
Sequence listing
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<120> a nicotinamide phosphoribosyltransferase mutant
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tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt ctggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gagccttatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 7
<211> 491
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Cys Gly Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Cys Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 8
<211> 1476
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt gcggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggactg cgttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 9
<211> 491
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Ser Cys Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Pro Val Cys Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 10
<211> 1476
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt cttgcaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agtttgcgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 11
<211> 491
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Pro Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Pro Glu Pro Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 12
<211> 1476
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt ctggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagaccct gagccttatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 13
<211> 491
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Cys Cys Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Cys Val Cys Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 14
<211> 1476
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt gctgcaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggactg cgtttgcgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 15
<211> 491
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Cys Cys Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Cys Val Cys Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Pro Glu Pro Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 16
<211> 1476
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt gctgcaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggacct gcttgtgcat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagaccct gagccttatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
Claims (5)
1. A nicotinamide phosphoribosyltransferase mutant is characterized in that single-point or multi-point mutation is generated at positions 314, 315, 417, 419, 450 and 452 by taking an amino acid sequence of a wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO.1 as a reference sequence. Wherein Ser at position 314 is mutated into Cys, Gly at position 315 is mutated into Cys, Pro at position 417 is mutated into Cys, Ala at position 419 is mutated into Cys, Leu at position 450 is mutated into Pro, and Glu at position 452 is mutated into Pro.
2. The nicotinamide phosphoribosyltransferase mutant of claim 1, wherein the amino acid sequence of the nicotinamide phosphoribosyltransferase mutant is shown in SEQ ID No.3, 5, 7, 9, 11, 13 and 15.
3. The nicotinamide phosphoribosyltransferase mutant of claim 1, wherein the nucleotide sequence of the gene of the ketoreductase mutant is shown in SEQ ID Nos. 4, 6, 8, 10, 12, 14 and 16.
4. The nicotinamide phosphoribosyltransferase mutant of claim 1, which is expressed in escherichia coli e.
5. The nicotinamide phosphoribosyltransferase mutant of claim 1, wherein the nicotinamide phosphoribosyltransferase mutant is capable of converting nicotinamide and phosphoribosyl pyrophosphate into β -nicotinamide mononucleotide.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108026517A (en) * | 2016-07-30 | 2018-05-11 | 邦泰生物工程(深圳)有限公司 | A kind of Nampt mutant and its application |
| CN108048420A (en) * | 2017-11-29 | 2018-05-18 | 天津市湖滨盘古基因科学发展有限公司 | The Nampt mutain of people a kind of and its application |
| CN109022383A (en) * | 2018-07-10 | 2018-12-18 | 天津市湖滨盘古基因科学发展有限公司 | The Nampt precursor mutain of people a kind of and its application |
| CN109666658A (en) * | 2018-12-27 | 2019-04-23 | 成都及禾生物科技有限公司 | It is used to prepare Nampt, encoding gene, recombinant vector and the application of NMN |
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2020
- 2020-09-10 CN CN202010945768.1A patent/CN114164189B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108026517A (en) * | 2016-07-30 | 2018-05-11 | 邦泰生物工程(深圳)有限公司 | A kind of Nampt mutant and its application |
| CN108048420A (en) * | 2017-11-29 | 2018-05-18 | 天津市湖滨盘古基因科学发展有限公司 | The Nampt mutain of people a kind of and its application |
| CN109022383A (en) * | 2018-07-10 | 2018-12-18 | 天津市湖滨盘古基因科学发展有限公司 | The Nampt precursor mutain of people a kind of and its application |
| CN109666658A (en) * | 2018-12-27 | 2019-04-23 | 成都及禾生物科技有限公司 | It is used to prepare Nampt, encoding gene, recombinant vector and the application of NMN |
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